Given the anticipated size of the data sets to be processed by the requested resource, the most efficient way to put data on and take it off is through hot swappable external 4Tb or 8Tb disk drives, a procedure requiring no technical expertise. The requested hardware has this capability. These drives currently cost about $20 per terabyte and would represent a modest expense for the Major Users. Minor users producing data via the Cryo-EM core already receive their data this way, so again, the requested facility will involve no new equipment or procedures.
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Instructions for Research Projects (30 pages max): In this section, describe the benefit of the requested instrument to enhance research projects. You can divide this section into subsections Research Projects of Major Users or Specific Research Topics. The latter format may be especially useful to avoid redundancies in the presentation of research projects if several Major Users pursue research topics which follow similar protocols and scientific benefits of the new instrument for their projects are comparable. All Major Users must have substantial need for the requested instrument. Detailed eligibility requirements for Major Users are described in Section III 3. In addition, if there are Minor Users and other users, include a subsection Minor Users’ Projects.
• Since the projects have been previously peer reviewed, describe their details only as necessary to explain how the requested instrument will advance the projects’ research objectives. (Do not simply copy the Specific Aims section from a funded application.) Present sufficient technical details about types of samples or specific experimental protocols to be employed to allow evaluation of whether the instrument is appropriate, would be effectively utilized, and would provide advantages over other methods and other similar existing or new instruments. In particular, explain the need for special features and accessories of the requested instrument by describing the specific studies that will utilize these options as at least three Major Users must need any of these special options. Preliminary data are not required, but if available, they may be used to illustrate the benefit of the requested instrument to the research projects. Describe how generated data will be handled and analyzed so that benefits of the entire experimental set-up can be judged. Summarize benefits that the requested instrument will provide towards answering specific scientific questions. Be succinct and clear.
• If you choose to divide this section into Research Projects of Major Users subsections, list the PD/PI’s name and grant information (number, title, project start and end dates) in the beginning of each subsection.
• If you choose to group research projects in subsections Specific Research Topics, in the beginning of each subsection list Major Users, their funded grants that you describe therein, and their cumulative usage as measured by the percentage of the AUT.
• Conclude this Research Projects section with a subsection Minor Users’ Projects to describe the need of the requested instrument to advance projects from Minor Users and the user community at your institution (e.g., unfunded users who have significant need for the instrument to develop their research programs or users whose expected needs are at the level of 1% or less of AUT).
• In cases of certain technologies (such as computer systems or X-ray detectors), a large number of users, exceeding what is necessary to make a strong case for the need of the instrument, may be expected. In such cases, you may select a representative smaller group of Major Users and describe their research projects’ needs in detail in subsections Research Projects of Major Users. Then, devote a separate subsection Other Users' Projects to describe research and instrumentation needs of your large user community, including Minor Users’. Keep in mind that the sole number of users is not a compelling factor to justify scientific needs for the requested instrument.
• You must focus this Research Projects section on detailed explanation of how the requested instrument will advance research projects. Research projects may be drawn from a broad array of topics in basic science, translational investigation or clinical trials; in particular, research projects on advancements of technologies for the benefit of biomedical research may be included. Demonstrate that NIH-funded investigators will use the instrument at the level of at least 75% of AUT.
• Section Guidelines: As adapted from the ACSB, this section should include the following: This section should begin with a brief summary of the major-user group—the schools, departments, and universities involved—and should state the broad use and support the instrument within the research community. List the major users first (project descriptions of two to three pages), then minor users (abbreviated project descriptions – one paragraph each is enough). Each research project should be organized as follows
1. PI name and title, PI role, and project title 2. One to three specific aims
3. Background and significance
4. Preliminary results that validate the need, use, and application of the requested equipment.
5. Experimental procedures and protocols to demonstrate your understanding of the use of the instrument and potential difficulties.
6. Use, application, and need for the requested instrument (including any accessories and unique capabilities) in fulfilling specific aims.
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Return to Section Instructions – Research Projects Return to Section Guidelines – Research Projects Continue on to Research Projects - Goldman Example Goldman –Research Projects
Major User Research Project
C.1.b. Axelsen, Paul H., Professor of Pharmacology Grant Numbers and Titles:
R01-GM076201, Structure Determination by Vibrational Spectroscopy R01-NS074178, Oxidative Lipid Stress in the Brain
Alzheimer’s disease is characterized by an as-yet-undefined process occurring in the vicinity of amyloid plaques that causes neuronal dysfunction and death. These plaques consist largely of amyloid β (Aββββ) peptides that have aggregated into fibrils. Despite intense study, the structure of these fibrils and the factors that induce their formation are unknown. However, it clear that the fibrils we prepare in vitro vary widely in molecular structure, as well as in their thermodynamic stability, and that fibril structure can “mature” over time.1
Project: The dye-binding mode of amyloid fibrils and their basis for specificity
We are currently engaged in a multi-faceted study of the way in which supposedly amyloid-specific fluorescent dyes bind to amyloid fibrils – with less-than-ideal instrumentation. There is more belief than data on this topic, and more long extrapolations from model systems than solid data obtained directly from amyloid fibrils. The role of MFD-FRET in this project is to determine the orientation of bound (immobilized) acceptor fluorescent dye molecules with respect to freely rotating donor fluorophores on the fibril axis. The orientation of the plane of the acceptor (parallel to the fibril axis, or perpendicular) will have a profound effect on its rotational rate, which should be straightforward to measure with MFD-FRET. The question of
orientation is important because reliable and detailed molecular structures are not available for fibrils, yet amyloid-specific binding is the basis of diagnostic imaging tests for Alzheimer’s disease. The repeating nature of amyloid fibril structure means that knowing whether dyes bind perpendicular or longitudinal offers important insight into what elements on the fibril amyloid-specific dyes are so specifically recognizing. With present instrumentation, we are limited by sensitivity and contrast against background because of sample requirements.
Specific Benefits of the Proposal/Equipment: Multiparameter fluorescence detection single-molecule FRET instrumentation would be an invaluable enhancement of our capability in these investigations because the range over which MFD-FRET measurements are informative (10-40 Å) is ideal for answering these questions, and far better than currently available instrumentation in which our samples are immobilized and subject to overwhelming amounts of background fluorescence. The current project will be much more elegantly performed by exciting bound dye molecules with donor fluorophores attached to the fibril, since that would dramatically reduce signals from nonspecifically excited background fluorescence. We routinely prepare fibril
“seeds” that embody all of the structural features of full-length amyloid fibrils. With dimensions that are roughly 10 nm in diameter and 100 nm long, they readily diffuse in solution,2-8 and bound fluorophores will exhibit markedly different rotational rates depending on their orientation relative to the long axis, detectable using the polarization and anisotropy capability of the instrument.
References
1. Ma,J.Q., Komatsu,H., Kim,Y.S., Liu,L., Hochstrasser,R.M. & Axelsen,P.H. Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid Beta Peptide Fibrils. ACS Chem.Neurosci.
4:1236-1243 (2013)
2. Watts,J.C., Condello,C., Stõhr,J., Oehler,A., Lee,J., DeArmond,S.J., Lannfelt,L., Ingelsson,M., Giles,K. &
Prusiner,S.B. Serial Propagation of Distinct Strains of Aβ Prions From Alzheimer's Disease Patients.
Proc.Natl.Acad.Sci.USA 111:10323-10328 (2014)
3. Stõhr,J., Condello,C., Watts,J.C., Bloch,L., Oehler,A., Nick,M., DeArmond,S.J., Giles,K., DeGrado,W.F. &
Prusiner,S.B. Distinct Synthetic Aβ Prion Strains Producing Different Amyloid Deposits in Bigenic Mice.
Proc.Natl.Acad.Sci.USA 111:10329-10334 (2014)
4. Lu,J.X., Qiang,W., Yau,W.M., Schwieters,C.D., Meredith,S.C. & Tycko,R. Molecular Structure of Beta-Amyloid Fibrils in Alzheimer's Disease Brain Tissue. Cell 154:1257-1268 (2013)
5. Petkova,A.T., Leapman,R.D., Guo,Z., Yau,W.-M., Mattson,M.P. & Tycko,R. Self-Propagating, Molecular-Level Polymorphism in Alzheimer's Β-Amyloid Fibrils. Science 307:262-265 (2005)
6. Kim,Y.S., Liu,L., Axelsen,P.H. & Hochstrasser,R.M. Two-Dimensional Infrared Spectra of Isotopically Diluted Amyloid Fibrils From Aβ40. Proc.Natl.Acad.Sci.USA 105:7720-7725 (2008)
7. Kim,Y.S., Liu,L., Axelsen,P.H. & Hochstrasser,R.M. 2D IR Provides Evidence for Mobile Water Molecules in Β-Amyloid Fibrils. Proc.Natl.Acad.Sci.USA 17751-17756 (2009)
8. Komatsu,H., Feingold-Link,E., Sharp,K.A., Rastogi,T. & Axelsen,P.H. Intrinsic Linear Heterogeneity of Amyloid Beta Protein Fibrils Revealed by Higher Resolution Mass-Per-Length Determinations.
J.Biol.Chem. 285:41843-41851(2010) Minor User Research Project
C.2.a. Deutsch, Carol, Professor of Physiology Grant Number and Title:
R01-GM052302 Biogenesis of Voltage-Gated K+ Channels
Project: Peptide Folding in the Ribosome Exit Tunnel
Protein synthesis involves a 2-way dynamism between the nascent peptide being elongated in the ribosome and the ribosome’s exit tunnel (Fig. 16). This specialized microenvironment is a tight squeeze for a nascent peptide, and it likely contains sensors and signaling mechanisms for peptide folding. We have found diverse functional zones along the tunnel1, 2, 3 and that relocation and/or reorientation of the nascent peptide (both short-range and long-range) relative to the tunnel depends on the nature of the primary sequence of the nascent peptide3. We suggest that these discoveries reflect a multiplicity of peptide conformations and trajectories, which underlie signaling between different tunnel regions during translation. The multi-parameter fluorescence detection of FRET signals will allow identification and analysis of individual species present in our pool of nascent peptides attached to the ribosome, with a time-resolution amenable to translation events and transit through the tunnel.
Using MFD-FRET, we will initially i). test the hypothesis that different nascent peptides move along different tunnel pathways, ii). test the hypothesis that secondary structure of a nascent peptide in the tunnel can be recon-figured by its emergent N-terminus, and iii). determine the nature of putative ‘compact’ (helical?) structures in different regions of the tunnel.
To this end, the optimal fluorophores for intramolecular MFD-FRET can be covalently coupled to our nascent peptides either via our arsenal of strategically engineered cysteines1, 3, 4 or through our synthesis and in-corporation of unnatural amino acids (Po and Deutsch, unpub. data). Fluorescently-tagged residues are ac-commodated in the ribosomal tunnel for ensemble FRET measurements5 and probe-tagged cysteines easily transit the tunnel during translation and fold correctly1, 3, 6.
Specific Benefits of the Proposal/Equipment: The proposed single molecule FRET instrument will be important to advance these studies because we expect significant heterogeneity among partly translated peptides and a given peptide sequence may have a distribution of pathways. Ensemble FRET measurements cannot resolve these possible natural variations in the population. Distance measurements that delineate the peptide secondary structures within the exit tunnel and thereby folding during translation need to be accurate to make clear interpretations. The capability of the MFD-FRET instrument requested to quantify probe mobility, relative probe orientation, donor lifetime and quantum yield during individual measurements, distributions of these variables, and associated analysis software are essential for obtaining quantitatively reliable, calibrated distances be-tween the labeled residues. The MFD-FRET measurements will thus complement and extend our unique and ongoing studies of co-translational folding.
References
1. Lu,J., Hua,Z., Kobertz,W.R., & Deutsch,C. Nascent peptide side-chains induce rearrangements in distinct locations of the ribosomal tunnel. J. Mol. Biol. 411, 499 -510 (2011).
2. Lu,J. & Deutsch,C. Folding zones inside the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 12, 1123-1129 (2005).
3. Lu,J. & Deutsch,C. Regional discrimination and propagation of local rearrangements along the ribosomal exit tunnel. J. Mol. Biol. 426, 4061-4073 (2014).
4. Tu,L., Khanna,P., & Deutsch,C. Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome. J. Mol. Biol. 426, 185-198 (2014).
5. Woolhead,C.A., McCormick,P.J., & Johnson,A.E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725-736 (2004).
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Research Projects – Gupta Example C1. Overview
The Optima AUC will support and immediately impact a diverse array of research projects from 8 major and 4 minor user groups that are all federally funded via the NIH (including NIGMS and NIAID) or the NSF. As a resource at the JRFSBBC, the instrument will not only serve investigators at Penn, but will also support the research of NIH and NSF investigators from other institutions in the region and from across the country. The projects presented herein reflect our typical usage patterns and are representative of the kinds of projects we expect to accelerate with this new technology. We expect that from year-to-year, accessible user time (AUT) allocated for each project will vary as studies arrive to a conclusion and new users and projects are introduced.
For new users we reserve time (5% AUT) to help generate preliminary data for new grant applications.
It is expected that the many improvements in the new Optima AUC will have immediate impact on these ongoing research projects, including increased data density (via multiwavelength data collection, improved radial resolution, and faster scan time), enhanced signal-to-noise and higher wavelength and radial precision.
In many of the projects presented, the 8-position An50Ti rotor alongside enhanced scan rates will make it possible to probe multiple conditions simultaneously in a single run across a range of wavelengths. This will enhance detailed study of protein oligomerization and hetero-associations (i.e., protein with DNA, protein-protein interactions, protein-protein-ligand binding) across a large range of concentrations, leveraging different extinction coefficients at different wavelengths, leading to a dynamic range larger than is now possible with single wavelength absorption optics in the XL-A instrument. Most any project arriving at the new instrument will benefit immediately from this enhanced capacity.
A fundamental strength of the AUC method is its ability to resolve heterogenous mixtures, a strength that is enhanced by the new instrument’s optical resolution. The density and quality of the data combined with state-of-the-art data analysis methods like those in ULTRASCAN-III will provide the best statistical fits to data possible, allowing for small hydrodynamic differences between individual species to be resolved. This feature will be important to projects with great heterogeneity (e.g., total ribosome profiling in User Project #6), and systems with confounding self-association of component parts. These assorted instrument features together further enable the opportunity to perform multi-wavelength experiments, a dimension to the projects presented not previously realized. In the past four years, the feasibility of these approaches has been demonstrated (9-11, 51) and now the analysis has been implemented in the ULTRASCAN-III software suite.
All the user projects described herein are well-established in vitro and optically heterogenous systems that will uniquely benefit from the enhanced technology and emerging multiwavelength methods. A synopsis of the types of projects presented are provided:
Table C1: Project Categories to be examined by Multiwavelength AUC.
Project Category Project User Status
I. Protein-Nucleic Acid complexes Gregory Van Duyne Major
Ben Black Major
Rahul Kohli Major
Kristin Lynch Major
Fange Liu Major
Lydia Contreras Major
II. Integral Membrane Proteins Vera Moiseenkova Minor
Fevzi Daldal Minor
III. Protein-Protein Interactions Yale Goldman Major Frederic Bushman and Gregory
Van Duyne Major
Eileen Jaffe Minor
Elizabeth Rhoades Minor
Key to the application of the multiwavelength approach and successful spectral deconvolution of the data are spectral absorbance profiles unique to the different species to be resolved. In the absence of specific labelling strategies or unique chromophores, it is expected to be difficult to resolve different proteins based on the ratio of their aromatic residues to the peptide bond contributions in the low UV without a very large number of collected wavelengths during the experiment. A major feature of all the user projects in this proposal are distinguishing optical features in the mixtures to be studied, including protein vs nucleic acid, heme chromophores, and labelled species with unique absorbance properties in complex mixtures. All these projects are already well positioned for immediate acceleration of ongoing research by the availability of the Optima instrument. In all cases, expression and purification schemes are well-established with quantities at the levels needed to support AUC study, and in all but one case, preliminary data has been collected using the older XL/A instrument or via pilot experiments on an Optima instrument located in Canada (the Demeler research group). And for many of the user projects described, labelling protocols have already worked out and applied.
Integral Membrane Proteins. The experimental systems described in Minor User Projects #2 and #3 (Daldal and Moiseenkova-Bell) are representative for a large and important class of systems involving integral membrane proteins (over a third of the human genome and among the most important drug targets).
Membrane-bound and associated proteins (including trans-membrane proteins, receptors, and channels) are often insoluble in aqueous medium, and therefore tend to aggregate without a surrogate carrier which emulates their native hydrophobic environments, such as detergents or lipid. The application of MW-AUC to their study provides an important complement to their structural biology, including the ongoing cryo-EM efforts at Penn, as the information gathered from these analyses are paramount to project success. These measurements provide rigorous quality control and valuable information for interpretation of samples undergoing structural analysis, including mass, shape, stoichiometry, and monodispersity.
Generally, such quality control is difficult to achieve with integral membrane proteins and cognate protein-detergent complexes due to technical challenges with conventional methods such as light scattering or
standard AUC methods. As needed, we will guide users to embed such proteins into nanodiscs to facilitate their study. Nanodiscs are protein-stabilized lipid rafts with are monodisperse and water-soluble. They can be used to mimic the native phospholipid bilayer to solubilize membrane-embedded targets. The size of the membrane bilayer in nanodiscs are stabilized by recombinant constructs of high-density apolipoprotein A1, which can be created in different lengths. Because nanodiscs are stable and their composition readily adjusted, this technology is a particularly powerful vehicle for the biophysical study of integral membrane proteins.
Nanodiscs will be prepared as described previously using established methods (52, 53) and are already
Nanodiscs will be prepared as described previously using established methods (52, 53) and are already